Cell functions affected by environmental unpredictability
J. Ignacio Lucas Lledó
23/1/2020
Introduction
On 2020-01-14 I run the analysis of Gene Ontology terms. Here I want to focus on part of those results and make some findings clearer. I will use results only from gene annotation, and not from annotation of transcripts, which is a somewhat noiser dataset. I will not look at the functions affected by hatching condition, since it only affects a couple of genes, to be inspected later. Among all cellular functions significantly enriched with genes differentially expressed among selective regimes, I will focus on those that are most significant, and detailed enough. For example, I skip terms like transmembrane transport, proteolysis or protein phosphorylation, because they are too general. Sections below are named for the functional categories that I think are worth discussing. I will try to contextualize the results. My entry point to the literature is (Garcia-Roger et al. 2019).
I need the results of the differential expression analysis (2020-01-08) to identify the genes annotated with the significant GO terms, and to assess the direction of gene expression change between selective regimes.
library(variancePartition)
library(GO.db)
library(ggplot2)
library(stringr)
ANNOTATION <- '../2019-07-26/genes/annotation.txt'
EXPRESSION <- '../2020-01-08/genes.RData' # variance partition and the mixed models fitted with dream()
INTERPROSCAN <- '../2019-07-19/functional_annotation.tsv' # annotation of transcripts
TRANS2GENE <- 'transcript2gene.txt' # created in this folder by README.sh from 2019-07-10/transcripts.faThe annotation table includes only the most specific terms that can be assigned to a gene. The more general, ancestor terms are assumed, of course, but not explicitly mentioned in the annotation. For the purpose of retrieving all genes annotated to a term, I need to expand the annotation, to make ancestor terms explicit. It took me a while to realize that I can do this with the unlist() function:
annotation <- read.table(ANNOTATION, col.names = c('gene', 'GOterms'), colClasses = c('character', 'character'))
head(annotation)## gene GOterms
## 1 XLOC_000002 GO:0008417|GO:0016020|GO:0006486
## 2 XLOC_000009 GO:0043130|GO:0005515|GO:0043161
## 3 XLOC_000010 GO:0005840|GO:0015935|GO:0003735|GO:0006412
## 4 XLOC_000015 GO:0006355|GO:0003700|GO:0006357|GO:0043565|GO:0005634|GO:0032502
## 5 XLOC_000021 GO:0016567|GO:0006397|GO:0061630|GO:0008270
## 6 XLOC_000036 GO:0005272|GO:0006814|GO:0016020annotation.list <- strsplit(annotation$GOterms, split='|', fixed=TRUE)
names(annotation.list) <- annotation$gene
# append can only join two vectors at a time. It works with lists!
allAncestors <- append(as.list(GOBPANCESTOR),
append(as.list(GOMFANCESTOR),
as.list(GOCCANCESTOR)))
fullAnnotation <- lapply(annotation.list,
FUN = function(x){
unique(append(x,
unlist(allAncestors[x], use.names=FALSE)))
})
load(EXPRESSION)Without any attempt to characterize gene function any further, I will make the assumption that the more genes in a pathway are expressed, the more active the pathway is. It is, however, conceivable that some genes are annotated as involved in a function that they regulate negatively.
Dormant or resting eggs are embryos in a dormant state. RNA was extracted either after forced diapause or not, but under hatching conditions. That is, a variable portion of eggs are expected to be leaving the dormancy stage and resuming metabolism, development and hatching. The portion of eggs getting ready to hatch is known to be larger (actually, a majority) in populations from regular environments. Thus, some of our results could resemble those from comparisons between resting and non-resting (amictic) eggs (Ziv et al. 2017, @Rozema2019).
We know that gene expression levels do not correlate with protein abundances (Ziv et al. 2017). In dormant embryos, where translation is downregulated (Ziv et al. 2017), this lack of correlation may be even more important. Many genes found to be expressed in dormant embryos may only be translated upon exit from dormancy.
# By using 'paste(goterm,collapse='|')', I can retrieve all genes annotated with any of a set of GO terms.
getGenes <- function(goterm, annotation = fullAnnotation, varpart = varPart) {
genes <- names(annotation[grep(paste(goterm, collapse='|'), annotation)])
if (! is.null(varpart)) {
genes <- genes[genes %in% row.names(varpart)]
}
return(genes)
}
volcanoPlot <- function(fit=fitmm, Coef='regime', num=length(fitmm$F), term=goterm, genes=getGenes(term)){
DE <- topTable(fit, coef=Coef, number=num)
DE$annotation <- factor('other', levels=c('other', Term(GOTERM[term])), ordered=TRUE)
DE[row.names(DE) %in% genes, 'annotation'] <- Term(GOTERM[term])
DE <- DE[order(DE$annotation),]
ggplot(data=DE, mapping=aes(x=logFC, y=-log10(P.Value), color=str_wrap(annotation, width=30))) + geom_point() +
labs(color='Annotation')
}For adequate interpretation of some categories, I will need to check the original functional annotation performed with interproscan on 2019-07-19. From one of the output tables, I select the most informative columns, potentially including names of functional domains detected in transcripts. I also need the correspondance between genes and transcripts, which is found in 2019-07-10/transcripts.fa.
# The file is tab-separated. Some rows miss the last two fields. Some e-values are '-'. And
# functional description strings may include the single quote character.
interpro <- read.table(INTERPROSCAN, sep='\t', fill=TRUE, na.strings=c('-',''), quote='"',
colClasses=c('character','NULL','integer','factor','character','character',
'NULL','NULL','numeric','NULL','NULL','character','character','character'),
col.names=c('transcript', NA, 'tsLength','analysis','acc','description', NA, NA,
'evalue', NA, NA,'IPacc','IPdescription','GO'))
interpro$transcript <- substr(interpro$transcript, 1, 14) # to remove the .p1, .p2... suffix.
ts2gene <- read.table(TRANS2GENE, colClasses=c('character','character'), col.names=c('transcript','gene'))
# Having all the information, I want a function that outputs the interpro annotations of a gene, a
# set of genes, or a GO term.
showFunctions <- function(gene, goterm=NULL, transcriptTable=ts2gene, annotation=interpro){
if (! is.null(gene)) {
return(annotation[annotation$transcript %in% transcriptTable[transcriptTable$gene %in% gene, 'transcript'],])
} else {
return(showFunctions(getGenes(goterm)))
}
}Biological processes
Nucleotide-excision repair
It is among the most significantly enriched terms, with only 13 genes annotated. In all volcano plots below, fold changes are computed with the expression level in the unpredictable regime in the numerator: if the log fold-change is negative, the expression is higher in the regular environment, and viceversa.
goterm <- 'GO:0006289'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
The expression of NER genes is higher in dormant embryos from populations evolved in the unpredictable environment. No idea why.
G protein-coupled receptor signaling pathway, and signal transduction
goterm <- 'GO:0007186'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Among the 206 genes in this category, 55 have an adjusted p-value lower or equal to 0.1. There are too many genes annotated with this function to plot all their stratified expression levels. I select the most differentially expressed ones.
Among the top 206 genes analysed from the G protein-coupled receptor signaling pathway, 173 have a reduced expression level in the unpredictable environment, and 33 show an increased expression level. Thus, in general we expect dormant eggs from populations under the unpredictable regime to be less sensitive to this particular pathway of signal transduction.
goterm <- 'GO:0007165'
genes <- getGenes(goterm)volcanoPlot()
544 genes are involved in signal transduction.
It seems that in general, signal transduction activity must be lower in resting eggs from populations under the unpredictable regime. This suggests that in unpredictable environments resting eggs rely less in (unreliable) environmental cues, and probably more in endogenous ones.
Cell-matrix adhesion
goterm <- 'GO:0007160'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Among the top 16 genes involved in cell-matrix adhesion, 14 have a reduced expression level in the unpredictable environment, and 2 show an increased expression level.
Cell-matrix adhesion is likely related to embryonic development.
Potassium ion transport, carboxylic acid transport, and ion transmembrane transport
Several functions related to ion transport are significant. I wonder to what extent their significance is not dependent on a common subset of genes.
goterm_K <- 'GO:0006813'
goterm_COOH <- 'GO:0046942'
goterm_Sulf <- 'GO:0008272'
goterm_Nucl <- 'GO:1901642'
goterm_TM <- 'GO:0034220'
genes_K <- names(fullAnnotation[grep(goterm_K, fullAnnotation, fixed=TRUE)])
genes_COOH <- names(fullAnnotation[grep(goterm_COOH, fullAnnotation, fixed=TRUE)])
genes_Sulf <- names(fullAnnotation[grep(goterm_Sulf, fullAnnotation, fixed=TRUE)])
genes_Nucl <- names(fullAnnotation[grep(goterm_Nucl, fullAnnotation, fixed=TRUE)])
genes_TM <- names(fullAnnotation[grep(goterm_TM, fullAnnotation, fixed=TRUE)])
vennDiagram(vennCounts(cbind(Potassium = row.names(varPart) %in% genes_K,
Carboxilic = row.names(varPart) %in% genes_COOH,
Sulfate = row.names(varPart) %in% genes_Sulf,
Nucleoside = row.names(varPart) %in% genes_Nucl,
Transmembrane = row.names(varPart) %in% genes_TM)))
There is hardly any overlap among genes involved in the transport of these substances. The only remarkable overlap happens between potassium ion transport and ion transmembrane transport. But, still the significance of each term should be quite independent of the significance of any other term. This was expected, because the analysis of functional enrichment applied algorithms to account for the dependence structure among GO terms. I analyse them separately, below.
In summary, most genes involved in ion transport, and also those involved in nucleoside transport, are expressed at lower levels in resting eggs from populations submitted to the random regime.
Potassium ion transport
goterm <- goterm_K
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Ion transmembrane transport
goterm <- goterm_TM
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Nucleoside transmembrane transport
goterm <- goterm_Nucl
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Carboxylic acid transport
goterm <- goterm_COOH
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Trehalose biosynthetic process
Trehalose is a disaccharide that confers protection against dessication in several organisms (n.d.). Trehalose is present in higher concentrations in resting eggs of B. plicatilis (which resist dessication) than in asexual eggs (Rozema et al. 2019). While trehalose is a very relevant molecule for dormant embryos, it is unclear why genes involved in its biosynthesis are expressed more in eggs from regular than from unpredictable environments.
goterm <- 'GO:0005992'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Cell motility, cilium movement and cilium assembly
Adult individuals of B. plicatilis have a crown of cilia. Thus, the expression of genes for cilium assembly, and probably also those for cilium movement and cell motility in the embryo must be related to embryonic development. They are, thus a sign that the embryos are leaving the dormant state and resuming metabolism. Under this light, it makes sense that eggs from a regular environment express these genes more than eggs from the unpredictable environment, because most of the former must be getting ready to hatch.
Dr. Roger has actually observed embryo movement in many of the eggs from the predictable regime after 30 hours of exposure to hatching conditions (personal communication).
goterm_cellmoti <- 'GO:0048870' # cell motility
goterm_movement <- 'GO:0003341' # cilium movement
goterm_assembly <- 'GO:0060271' # cilium assembly
genes_cellmoti <- getGenes(goterm_cellmoti)
genes_movement <- getGenes(goterm_movement)
genes_assembly <- getGenes(goterm_assembly)
vennDiagram(vennCounts(cbind('Cell motility' = row.names(varPart) %in% genes_cellmoti,
'Cilium movement' = row.names(varPart) %in% genes_movement,
'Cilium assembly' = row.names(varPart) %in% genes_assembly)))
Cell motility
goterm <- goterm_cellmoti
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Cilium movement
goterm <- goterm_movement
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Cilium assembly
goterm <- goterm_assembly
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Proteasome-mediated ubiquitin-dependent protein catabolic process
goterm <- 'GO:0043161'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
It is unclear why preteasome-mediated protein degradation should be more or less active in one or the other regime. And the patter is actually not consistent: two genes from this pathway are quite significantly (but not very much) more expressed in the unpredictable than in the regular environment. But several other genes in the pathway are less significantly, but to a higher degree underexpressed in the unpredictable regime. A fruitful interpretation may require individual inspection of those genes.
z <- topTable(fitmm, coef='regime', number=length(fitmm$F), p.value=0.1)
z <- z[row.names(z) %in% genes,]
PosLFC <- row.names(z[z$logFC > 0,])
NegLFC <- row.names(z[z$logFC < 0,])
zRandom <- unique(c(showFunctions(PosLFC)[,'description'],
showFunctions(PosLFC)[,'IPdescription']))
zRegular <- unique(c(showFunctions(NegLFC)[,'description'],
showFunctions(NegLFC)[,'IPdescription']))Among the 16 most significantly differentially expressed genes presumably involved in the proteasome-mediated ubiquitin-dependent protein catabolic process, the 6 that are overexpressed in the unpredictable environment encode some protein domains that are not found among the 10 genes overexpressed in the regular regime; namely: WW, PIN_Swt1-like, WW domain, WW/rsp5/WWP domain profile., PIN domain, Ubl_TMUB1_like, Ubiquitin domain profile., Ubiquitin family, BBOX, ATP-dependent Clp protease adaptor protein ClpS, Putative zinc finger in N-recognin (UBR box), Zinc finger UBR-type profile., RING-CH-C4HC3_LTN1, Ring finger domain, Zinc finger RING-type profile., F-box-like, F-box domain profile., PIN-like domain superfamily, Ubiquitin-like domain superfamily, Ubiquitin domain, Transmembrane and ubiquitin-like domain-containing protein 1/2, B-box-type zinc finger, E3 ubiquitin-protein ligase UBR1-like, Adaptor protein ClpS, core, Ribosomal protein L7/L12, C-terminal/adaptor protein ClpS-like, Zinc finger, UBR-type, WW domain superfamily, Listerin, zinc finger, RING-type, Zinc finger, RING/FYVE/PHD-type, E3 ubiquitin-protein ligase listerin, Zinc finger, RING-type, Leucine-rich repeat domain superfamily, F-box domain, F-box-like domain superfamily.
Likewise, among the genes involved in this biological process that are overexpressed in the regular environment, we see the following protein domains encoded: Mucin-2 protein WxxW repeating region, VWFD domain profile., WxxW domain, von Willebrand factor, type D domain, Quinoprotein alcohol dehydrogenase-like superfamily, Soluble quinoprotein glucose/sorbosone dehydrogenase.
Response to oxidative stress
Oxidative stress is one of the threats faced by dormant embryos, and protection against it is one of the classic hallmarks of dormancy (Clark et al. 2012,@Ziv2017).
goterm <- 'GO:0006979'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
In this case again, the differential expression does not have the same sign in all genes from this category that are regulated by the selective regime.
z <- topTable(fitmm, coef='regime', number=length(fitmm$F), p.value=0.1)
z <- z[row.names(z) %in% genes,]
PosLFC <- row.names(z[z$logFC > 0,])
NegLFC <- row.names(z[z$logFC < 0,])
zRandom <- unique(c(showFunctions(PosLFC)[,'description'],
showFunctions(PosLFC)[,'IPdescription']))
zRegular <- unique(c(showFunctions(NegLFC)[,'description'],
showFunctions(NegLFC)[,'IPdescription']))Among the 26 annotated with the term for response to oxidative stress, only 12 are significantly regulated by the selective regime at 0.1 FDR: 4 upregulated in the unpredictable regime, and 8 upregulated in the regular regime. The protein domains encoded in the former but not in the latter are: Glutathione peroxidase family signature, Glutathione peroxidases signature 2., Glutathione peroxidase, Glutathione peroxidases active site., Glutathione peroxidase profile., TIGR00357: methionine-R-sulfoxide reductase, SelR domain, Methionine-R-sulfoxide reductase (MsrB) domain profile., GSH_Peroxidase, Glutathione peroxidase conserved site, Glutathione peroxidase active site, Thioredoxin-like superfamily, Peptide methionine sulphoxide reductase MrsB, Mss4-like superfamily, Peptide methionine sulfoxide reductase.
And found only in the genes overexpressed in the regular environment, we have: Protein kinase domain profile., AGC-kinase C-terminal domain profile., Protein kinases ATP-binding region signature., Protein kinase domain, Hr1 repeat, Serine/Threonine protein kinases active-site signature., HR1_PKN_2, REM-1 domain profile., HR1_PKN_1, Protein kinase-like domain superfamily, AGC-kinase, C-terminal, HR1 rho-binding domain, HR1 repeat superfamily, Protein kinase, ATP binding site, Serine/threonine-protein kinase, active site, Peroxidasin, Serine/threonine-protein kinase N, first HR1 domain.
Molecular functions
Motor activity
goterm <- 'GO:0003774'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
The three most significant genes here are overexpressed in eggs from the unpredictable environment, which seems to contrast the results for biological processes of cell motility and cilium movement. However, motor activity is defined as Catalysis of the generation of force resulting either in movement along a microfilament or microtubule, or in torque resulting in membrane scission, coupled to the hydrolysis of a nucleoside triphosphate.. Only 1 genes involved in motor activity is also involved in cilium movement.
Peptidase activities: metallocarboxypeptidase activity, metalloendopeptidase activity, serine-type endopeptidase activity and calcium-dependent cysteine-type endopeptidase activity
goterm_metallocarboxy <- 'GO:0004181'
goterm_metalloendopep <- 'GO:0004222'
goterm_serinetypeendo <- 'GO:0004252'
goterm_calciumcystein <- 'GO:0004198'
genes_metallocarboxy <- names(fullAnnotation[grep(goterm_metallocarboxy, fullAnnotation, fixed=TRUE)])
genes_metalloendopep <- names(fullAnnotation[grep(goterm_metalloendopep, fullAnnotation, fixed=TRUE)])
genes_serinetypeendo <- names(fullAnnotation[grep(goterm_serinetypeendo, fullAnnotation, fixed=TRUE)])
genes_calciumcystein <- names(fullAnnotation[grep(goterm_calciumcystein, fullAnnotation, fixed=TRUE)])
vennDiagram(vennCounts(cbind(Metallocarboxy. = row.names(varPart) %in% genes_metallocarboxy,
Metalloendo. = row.names(varPart) %in% genes_metalloendopep,
Serine_type = row.names(varPart) %in% genes_serinetypeendo,
Cystein_type = row.names(varPart) %in% genes_calciumcystein)))
Metallocarboxypeptidase activity
goterm <- 'GO:0004181'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Serine-type endopeptidase activity
goterm <- 'GO:0004252'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Calcium-dependent cysteine-type endopeptidase activity
goterm <- 'GO:0004198'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Metalloendopeptidase activity
goterm <- 'GO:0004222'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced ascorbate as one donor, and incorporation of one atom of oxygen
goterm <- 'GO:0016715'
genes <- getGenes(goterm)
plotPercentBars(varPart[genes[order(-varPart[genes, 'regime'])],
c('regime', 'treatment', 'population', 'regime:treatment', 'Residuals')])
volcanoPlot()
Session info
sessionInfo()## R version 3.6.2 (2019-12-12)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.4 LTS
##
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.7.1
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.7.1
##
## locale:
## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
## [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
## [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] stats4 parallel stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] stringr_1.4.0 GO.db_3.10.0 AnnotationDbi_1.48.0
## [4] IRanges_2.20.2 S4Vectors_0.24.3 variancePartition_1.16.1
## [7] Biobase_2.46.0 BiocGenerics_0.32.0 scales_1.1.0
## [10] foreach_1.4.7 limma_3.42.0 ggplot2_3.2.1
##
## loaded via a namespace (and not attached):
## [1] bit64_0.9-7 splines_3.6.2 gtools_3.8.1
## [4] assertthat_0.2.1 blob_1.2.1 yaml_2.2.0
## [7] progress_1.2.2 pillar_1.4.3 RSQLite_2.2.0
## [10] backports_1.1.5 lattice_0.20-38 glue_1.3.1
## [13] digest_0.6.23 minqa_1.2.4 colorspace_1.4-1
## [16] htmltools_0.4.0 Matrix_1.2-18 plyr_1.8.5
## [19] pkgconfig_2.0.3 purrr_0.3.3 gdata_2.18.0
## [22] lme4_1.1-21 BiocParallel_1.20.1 tibble_2.1.3
## [25] farver_2.0.3 withr_2.1.2 lazyeval_0.2.2
## [28] pbkrtest_0.4-7 magrittr_1.5 crayon_1.3.4
## [31] memoise_1.1.0 evaluate_0.14 doParallel_1.0.15
## [34] nlme_3.1-143 MASS_7.3-51.5 gplots_3.0.1.2
## [37] tools_3.6.2 prettyunits_1.1.0 hms_0.5.3
## [40] lifecycle_0.1.0 munsell_0.5.0 colorRamps_2.3
## [43] compiler_3.6.2 caTools_1.18.0 rlang_0.4.2
## [46] grid_3.6.2 nloptr_1.2.1 iterators_1.0.12
## [49] labeling_0.3 bitops_1.0-6 rmarkdown_2.1
## [52] boot_1.3-24 gtable_0.3.0 codetools_0.2-16
## [55] DBI_1.1.0 reshape2_1.4.3 R6_2.4.1
## [58] knitr_1.27 dplyr_0.8.3 bit_1.1-15.1
## [61] zeallot_0.1.0 KernSmooth_2.23-16 stringi_1.4.5
## [64] Rcpp_1.0.3 vctrs_0.2.1 tidyselect_0.2.5
## [67] xfun_0.12References
Clark, Melody S., Nadav Y. Denekamp, Michael A. S. Thorne, Richard Reinhardt, Mario Drungowski, Marcus W. Albrecht, Sven Klages, Alfred Beck, Michael Kube, and Esther Lubzens. 2012. “Long-Term Survival of Hydrated Resting Eggs from Brachionus plicatilis.” PLoS ONE 7 (1): e29365. https://doi.org/10.1371/journal.pone.0029365.
Garcia-Roger, Eduardo M., Esther Lubzens, Diego Fontaneto, and Manuel Serra. 2019. “Facing Adversity: Dormant Embryos in Rotifers.” The Biological Bulletin 237 (2): 119–44. https://doi.org/10.1086/705701.
Rozema, Evelien, Sylwia Kierszniowska, Oshri Almog-Gabai, Erica G. Wilson, Young Hae Choi, Robert Verpoorte, Reini Hamo, Vered Chalifa-Caspi, Yehuda G. Assaraf, and Esther Lubzens. 2019. “Metabolomics Reveals Novel Insight on Dormancy of Aquatic Invertebrate Encysted Embryos.” Scientific Reports 9: 8878. https://doi.org/https://doi.org/10.1038/s41598-019-45061-x.
Ziv, T., V. Chalifa-Caspi, N. Denekamp, I. Plaschkes, S. Kierszniowska, I. Blais, A. Admon, and E. Lubzens. 2017. “Dormancy in Embryos: Insight from Hydrated Encysted Embryos of an Aquatic Invertebrate.” Molecular and Cellular Proteomics 16 (10): 1746–69. https://doi.org/10.1074/mcp.RA117.000109.
n.d.